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The Oxygen Effect and Reoxygenation

The Oxygen Effect and Reoxygenation. Radiobiology for the Radiologist, chapter 6, pg 91 - 111. The Nature of the Oxygen Effect. The Nature of the Oxygen Effect. Survival curves for mammalian cells exposed to x-rays in the presence and absence of oxygen Sensitivity to x-rays Aerated → S↑

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The Oxygen Effect and Reoxygenation

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  1. The Oxygen Effect and Reoxygenation Radiobiology for the Radiologist, chapter 6, pg 91 - 111

  2. The Nature of the Oxygen Effect

  3. The Nature of the Oxygen Effect • Survival curves for mammalian cells exposed to x-rays in the presence and absence of oxygen • Sensitivity to x-rays • Aerated → S↑ • Hypoxia → S↓ • Oxygen enhancement ratio (OER) • The ratio of hypoxic to aerated doses needed to achieve the same biological effect • High dose (dose > 2Gy) • OER = 2.5 – 3

  4. The Nature of the Oxygen Effect • Low dose (dose < 2 Gy) • OER = 2 • Reasons: • Variation of OER with the phase of the cell cycle • OER (G1 phase) < OER (S phase) • G1 more radiosensitive • Dominate the survival at low dose region

  5. The Nature of the Oxygen Effect • The oxygen enhancement ratio (OER) for various types of radiation Large & important Intermediate Absent

  6. The Time at which Oxygen Acts

  7. The Time at which Oxygen Acts • Experiment for the time at O2 acts • O2 at high pressure chamber • “explode” onto single layer bacteria • At various time before or after irradiation • Result: • Oxygen need not be present during the irradiation to sensitize • Could be added afterward

  8. Mechanism of the Oxygen Effect

  9. Mechanism of the Oxygen Effect • Chain of events from the absorption of radiation Absorption of radiation Production of fast charged particles Passing through biologic material Production of a number of ion pairs (10–10 sec) Free radicals (10–5 sec) Break chemical bond Biological damage

  10. Mechanism of the Oxygen Effect • O2 acts at the level of the free radicals • Reacts with the free radical • R‧+ O2 → RO2‧ • Form organic peroxide • Oxygen fixation hypothesis • The damage produced by free radicals in DNA can be repaired under hypoxia • May be “fixed” if molecular oxygen is available

  11. The Concentration of Oxygen Required

  12. The Concentration of Oxygen Required • Survival curve for Chinese hamster cells exposed to x-rays in the presence of various oxygen concentrations • Result • 100 ppm, noticeable in change • 2200 ppm, halfway toward the fully aerated condition ○ Air ● 2200 ppm, 1.7 mmHg □ 355 ppm, 0.25 mmHg ■ 100 ppm, 0.075 mmHg △ 10 ppm, 0.0076 mmHg

  13. The Concentration of Oxygen Required • The dependence of radiosensitivity on oxygen concentration • Most of this change of sensitivity • Increase from 0 – 30 mmHg • Further increase • little further effect • Sensitivity halfway • pO2 : 3mm Hg

  14. The Concentration of Oxygen Required • Conclusion • Very small amounts of oxygen are necessary to produce the dramatic and important oxygen effect observed with x-rays • Oxygen tension of the body tissues • Venous blood or lymph → 20 – 40 mmHg • Different tissues may vary over a wide range from 1 – 100 mmHg • Borderline hypoxic tissue, e.g. liver, skeletal muscle

  15. Chronic Hypoxia

  16. Chronic Hypoxia • Definition • Result from the limited diffusion distance of oxygen through tissue that is respiring • First described by Thromlison and Gray • Specimen : bronchial carcinoma • Cell of the stratified squamous cell carcinoma

  17. Transverse section of tumor cord. Stroma Surrounded by intact tumor cells Central necrosis A typical tumor area in which necrosis is not far advanced.

  18. Bands of tumor cells Stroma Large areas of necrosis Large areas of necrosis separated from the stroma by bands of tumor cells about 100 mm wide

  19. Chronic Hypoxia • The conclusion • Small tumor cord • Radius < 160 μm • No necrosis • Tumor cord > 200 μm • Present of necrotic center • Tumor cord enlarged further • Thickness of the sheath of viable tumor cells remained essentially constant (100 – 180 μm)

  20. The diffusion of oxygen from a capillary through tumor tissue ★ ★ • O2 is high enough for the • cells to be viable • O2 is low enough for them • to be relative protected • from the effects of x-rays • These cells may limit the • radiocurability of the • tumor • Proposed solution • High pressure oxygen • chamber • Neutrons • Negative π-mesons • Heavy charged ions

  21. Acute Hypoxia

  22. Acute Hypoxia • Definition • Develop in tumors as a result of the temporary closing or blockage of a particular blood vessel • Tumor blood vessels open and close in a random fashion • Different regions of the tumor become hypoxic intermittently • First postulated by Brown in 1980s

  23. Acute Hypoxia • Result from • temporary • closing of tumor • blood vessels • The cells are • intermittently • hypoxic • Normoxia is • restored each • time the blood • vessel opens up • again Acute hypoxia Chronic Hypoxia Diagram illustrating the difference between chronic and acute hypoxia

  24. The First Experimental Demonstration of Hypoxic cells in a tumor

  25. The First Experimental Demonstration of Hypoxic cells in a tumor • By Powers and Tolmach • Technique: dilution assay technique • Aim: investigate the radiation response of a solid subcutaneous lymphosarcoma in the mouse • Survival estimates: between 2 – 20 Gy

  26. First component Dose : < 9 Gy Slope (D0) : 1.1 Gy Second component Dose > 9 Gy Slope (D0) : 2.6 Gy 2.5 time shallower Fraction of surviving cells as a function of dose for a solid subcutaneous lymphosarcoma in the mouse irradiated in vivo

  27. The First Experimental Demonstration of Hypoxic cells in a tumor • The survival curve consists of two separate component • Strongly suggests that the tumor consist of two separate groups of cells • Oxygenated cells • Hypoxic cells • The shallow component of the curve cut the surviving fraction axis • Survival level : 1% • Means: 1% of the clonogenic cells in the tumor were deficient in O2

  28. The First Experimental Demonstration of Hypoxic cells in a tumor • At lower doses • dominated by the killing of the well-oxygenated cells • At higher doses • Oxygenated cells are depopulated severely • The response of the tumor is characteristic of the response of hypoxic cells • Conclusion • A solid tumor could contain cells sufficiently hypoxic to be protected from cell killing by x-rays • Still clonogenic and capable for tumor regrowth

  29. Proportion of Hypoxic cells in Various Animal Tumors

  30. Proportion of Hypoxic cells in Various Animal Tumors • Moulder and Rockwell • Published a survey of all published data in hypoxic fractions • 42 tumor types studies • 37 tumor types contain hypoxic cells • Hypoxic fraction: • range from 0 – 50% • Average: about 15% • Dische and Denekamp • Proportion of hypoxic cells in human • Consistence with the 10 – 15 % characteristic of many animal tumors

  31. Evidence for Hypoxia in Human Tumors • Analogy can be made with mouse tumors, in which hypoxia can be demonstrated unequivocally. • Histologic appearance suggests the possibility of hypoxia • Blinding of radioactive-labeled nitroimidazoles occurs • Oxygen-probe measurements are predictive • Pretreatment hemoglobin levels are powerful prognostic factor in SCC of the cervix, carcinoma of the bronchus, and TCC of the bladder

  32. Reoxygenation

  33. Reoxygenation • Van Putten and Kallman • Determined the proportion of hypoxic cell in mouse sarcomawithout irradiation and after various fractionated radiation treatment. • Result: • Proportion of hypoxic cells • Untreated : 14% • 1.9 Gy/Fx/Day × 5 days : 18% (test in 3 days later) • 1.9 Gy/ Fx/Day × 4 days : 14% (test in 1 day later) • The proportion of hypoxic cells is about the same.

  34. Reasons: • A dose of x-rays kills a greater proportion of aerated than hypoxic cells • More radiosensitive • After oxygenation, preirradiation pattern tends to return • Significant of fractionation • Allow sufficient time for oxygenation • The presence of hypoxic cells does not greatly influence the response of tumor

  35. Time Sequence of Reoxygenation

  36. Time Sequence of Reoxygenation • Percentage of hypoxic cells in a transplantable mouse sarcoma as a function of time after a dose of 10 Gy of x-rays • By Kallman & Bleehen Immediately after irradiation 100% of viable cells are hypoxic By 6 hours, percentage of hypoxic cells has fallen to a close value to the preirradiation level

  37. The extent and rapidity of reoxygenation is extremely variable and impossible to predict △Mouse osteosarcoma ● mouse fibrosarcoma ▲Rat sarcoma ○Mouse mammary carcinoma The proportion of hypoxic cells as a function of time after irradiation with a large dose for 5 transplanted tumors in experimental animals

  38. Mechanism of Reoxygenation • In chronic hypoxia • Cell killed by radiation are broken down and removed from tumor population • Restructuring or a revascularization of tumor • Tumor shrinks in size • Closer to blood supply • Taking place over period of days as the tumor shrinks • In acute hypoxia • Blood vessel is temporarily closed during irradiation • Quickly reoxygenate when that vessel reopens

  39. The Important of Reoxygenation in Radiotherapy

  40. The Important of Reoxygenation in Radiotherapy • The reoxygenation studies with C3H mouse mammary carcinoma • Reoxygenation 2 – 3 days after irradiation • The proportion of hypoxic cells is lower than in untreated tumors • Prediction • Several large dose of x-rays given at 48 hours intervals would virtually eliminate the problem of hypoxic cells in this tumor

  41. The Important of Reoxygenation in Radiotherapy • Fowler and his colleagues • The x-ray schedule for cure of this tumor • Five large doses in 9 days • Suggestion • X-irradiation can be an extremely effective form of therapy • But ideally required optimal choice of fractionation pattern

  42. The Important of Reoxygenation in Radiotherapy • Demands a detailed knowledge of the time course of reoxygenation in the particular tumor to be irradiated • Available for only a few animal tumors • Impossible to obtain for human • Evidence from radiotherapy clinic • Eradication doses for many tumors • 60 Gy in 30 fractions • Hypothesis • Human tumors do not respond to conventional R/T • Do not reoxygenate quickly and efficiently

  43. Hypoxia and Tumor Progression

  44. Hypoxia and Tumor Progression • Clinical study in Germany • Correlation between local control in advance carcinoma of the cervix, treated by R/T • Using O2 probe measurement • Result • pO2s > 10 mmHg → local control↑ • pO2s < 10 mmHg → local control↓ • Suggestion • Hypoxia is a general indicator of tumor aggression

  45. Hypoxia and Tumor Progression • Another study in United State • Soft-tissue sarcoma for R/T • Correlation between tumor oxygenation and the frequency of distant metastases • Result • pO2s > 10 mmHg → distant metastasis 35 % • pO2s < 10 mmHg → distant metastasis 70 % • Conclusion • Level of tumor oxygenation influences the aggressiveness of the tumor

  46. Hypoxia and Tumor Progression Inactivation → p53 tumor suppression gene Overexpression → bcl-2 antiapoptotic gene Illustration how hypoxia is linked with malignant progression

  47. The End Thanks for your attention !

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